Methods of depleting a target molecule from an initial collection of nucleic acids, and compositions and kits for practicing the same

ABSTRACT

Provided are methods of depleting a target nucleic acid from an initial collection of nucleic acids. Aspects of the methods include contacting the initial collection with a nucleic acid guided nuclease specific for the target nucleic acid in a manner sufficient to deplete the target nucleic acid from the initial collection. Depending on a given application, depletion of a target nucleic acid may vary, e.g., where depleting may include cleaving a target nucleic acid in, or selectively separating a target nucleic acid from, the initial collection of nucleic acids. Also provided are compositions and kits for practicing embodiments of the methods.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/582,081, filed Dec. 23, 2014, now U.S. Pat. No. 10,150,985; whichclaims priority to U.S. Provisional Patent Application Nos. 61/939,658,filed Feb. 13, 2014, and 62/040,804, filed Aug. 22, 2014; the aboveapplications are herein incorporated by reference.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The Sequence Listing is concurrently submitted herewith with thespecification as an ASCII formatted text file via EFS-Web with a filename of Sequence Listing.txt with a creation date of Oct. 20, 2016, anda size of 9.22 kilobytes. The Sequence Listing filed via EFS-Web is partof the specification and is hereby incorporated in its entirety byreference herein.

INTRODUCTION

Applications in biomedical research often involve the analysis ofspecific subsets of nucleic acids present in a complex mixture of othersequences—for example, analysis of gene expression by arrayhybridization, qPCR or massively parallel sequencing. If the sequencesof nucleic acids of interest are known, PCR with specific primersequences can be used to amplify the desired sequences out of themixture. In some cases, however, it may be desired to analyze multipledifferent sequences, perhaps where sequence information is not fullyknown. Messenger RNAs in eukaryotic systems, for example, may becollectively amplified and analyzed using an oligo-dT primer to initiatefirst strand cDNA synthesis by priming on the poly A tail, therebyreducing or avoiding contamination by unwanted nucleic acids—such asribosomal RNAs, mitochondrial RNAs and genomic DNA. A requirement forthis approach, however, is that the RNA is intact and not degraded,e.g., the poly A tails are not lost or disconnected from the body of theRNA message. Unfortunately, many otherwise useful and interestingbiological specimens—such as biopsied material retained asformalin-fixed and paraffin embedded tissue samples (FFPE samples) oftensuffer from such degradation making oligo-dT priming impractical forsuch samples. Further, many interesting RNA sequences do not have poly Atails—e.g., non-coding RNAs and non-eukaryotic RNAs. In such cases,random priming can be used to generally amplify all nucleotide speciesin the sample. However, random priming will also result in theamplification of potentially unwanted sequences—such as genomic DNA orribosomal RNA.

SUMMARY

Provided are methods of depleting a target nucleic acid from an initialcollection of nucleic acids. Aspects of the methods include contactingthe initial collection with a nucleic acid guided nuclease specific forthe target nucleic acid in a manner sufficient to deplete the targetnucleic acid from the initial collection. Depending on a givenapplication, depletion of a target nucleic acid may vary, e.g., wheredepleting may include cleaving a target nucleic acid in, or selectivelyseparating a target nucleic acid from, the initial collection of nucleicacids. Also provided are compositions and kits for practicingembodiments of the methods.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 schematically illustrates a nucleic acid guided nuclease thatfinds use in certain embodiments of the present disclosure (SEQ ID NOs:40-42).

FIG. 2 schematically illustrates a method for producing a nucleic acidguide component according to one embodiment of the present disclosure.

FIG. 3 shows three examples of oligonucleotides (T7-T1-AcGFP-SEQ ID NO:43; T7-T2-AcGFP-SEQ ID NO: 44; T7-Rev-SEQ ID NO: 45) that find use inthe method schematically illustrated in FIG. 2.

FIG. 4 shows an image of template nucleic acids visualized by gelelectrophoresis. The template nucleic acids find use in producingnucleic acid guide components by in vitro transcription according to oneembodiment of the present disclosure.

FIG. 5 shows an image of nucleic acids visualized by gelelectrophoresis. The image demonstrates the cleavage of a target nucleicacid using a nucleic acid guided nuclease according to one embodiment ofthe present disclosure.

FIG. 6 shows an image of nucleic acids visualized by gelelectrophoresis. The image demonstrates the simultaneous cleavage of twodifferent target nucleic acids using nucleic acid guided nucleasesaccording to one embodiment of the present disclosure.

FIG. 7 provides data demonstrating the depletion of a target nucleicacid (18S rRNA in this example) in a nucleic acid library for nextgeneration sequencing using a nucleic acid guided nuclease according toone embodiment of the present disclosure. Panel A shows a bar graphindicating the amount of depletion of a target nucleic using one or twoexample nucleic acid guided nucleases. Panel B shows the amount ofdepletion of the target nucleic acid using increasing amounts of anuclease according to one embodiment of the present disclosure. In thisexample, a pool of nucleic acid guided nucleases is employed, in whichthe pool includes a single type of nuclease component and variousspecies of nucleic acid guide components having different nucleotidesequences.

FIG. 8 provides data demonstrating the depletion of a target nucleicacid using a nucleic acid guided nuclease according to one embodiment ofthe present disclosure. In this example, a pool of nucleic acid guidednucleases is employed, in which the pool includes a single type ofnuclease component and various species of nucleic acid guide componentshaving different nucleotide sequences.

FIG. 9 is a bar graph showing sequencing results and demonstrating theeffects of depleting a target nucleic acid from a sequencing libraryusing a method according to one embodiment of the present disclosure.

FIG. 10 provides data demonstrating the cleavage of a target nucleicacid using a nucleic acid guided nuclease according to one embodiment ofthe present disclosure. Panel A shows a gel image demonstrating thepurification of an example nuclease component according to oneembodiment of the present disclosure. Panel B shows a gel imagedemonstrating the cleavage of a target nucleic acid using a nucleic acidguided nuclease that includes the nuclease component shown in Panel A.

FIG. 11 shows a gel image demonstrating cleavage of a target nucleicacid using various amounts of a nucleic acid guided nuclease accordingto one embodiment of the present disclosure.

FIG. 12 provides data demonstrating the expression and purification of a6×HN tagged D10A/H840A mutant of Cas9 (Panel A), and proof-of-principleof the use of the mutant in combination with a pool of nucleic acidguide components to remove target nucleic acids from an initialcollection of nucleic acids and produce a nucleic acid sample enrichedfor the target nucleic acids (Panel B).

DETAILED DESCRIPTION

Provided are methods of depleting a target nucleic acid from an initialcollection of nucleic acids. Aspects of the methods include contactingthe initial collection with a nucleic acid guided nuclease specific forthe target nucleic acid in a manner sufficient to deplete the targetnucleic acid from the initial collection. Depending on a givenapplication, depletion of a target nucleic acid may vary, e.g., wheredepleting may include cleaving a target nucleic acid in, or selectivelyseparating a target nucleic acid from, the initial collection of nucleicacids. Also provided are compositions and kits for practicingembodiments of the methods.

Before the methods and kits of the present disclosure are described ingreater detail, it is to be understood that the methods and kits are notlimited to particular embodiments described, as such may, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to be limiting, since the scope of the methods and kits will belimited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the methods and kits. The upper andlower limits of these smaller ranges may independently be included inthe smaller ranges and are also encompassed within the methods and kits,subject to any specifically excluded limit in the stated range. Wherethe stated range includes one or both of the limits, ranges excludingeither or both of those included limits are also included in the methodsand kits.

Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the methods belong. Although any methods and kitssimilar or equivalent to those described herein can also be used in thepractice or testing of the methods and kits, representative illustrativemethods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods, kits and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present methods and kits are not entitled to antedatesuch publication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

It is appreciated that certain features of the methods and kits, whichare, for clarity, described in the context of separate embodiments, mayalso be provided in combination in a single embodiment. Conversely,various features of the methods and kits, which are, for brevity,described in the context of a single embodiment, may also be providedseparately or in any suitable sub-combination. All combinations of theembodiments are specifically embraced by the present invention and aredisclosed herein just as if each and every combination was individuallyand explicitly disclosed, to the extent that such combinations embraceoperable processes and/or devices/systems/kits. In addition, allsub-combinations listed in the embodiments describing such variables arealso specifically embraced by the present methods and kits and aredisclosed herein just as if each and every such sub-combination wasindividually and explicitly disclosed herein.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentmethods and kits. Any recited method can be carried out in the order ofevents recited or in any other order which is logically possible.

In further describing embodiments of the invention, aspects ofembodiments of the subject methods will be described first in greaterdetail. Thereafter, aspects of embodiments of the kits for practicingthe subject methods are described in greater detail.

Methods

Aspects of the invention include methods of selectively depleting atarget nucleic acid from an initial collection of nucleic acids. By“depleting” a target nucleic acid is meant reducing the amount of thetarget nucleic acid in the initial collection of nucleic acids. Forexample, a target nucleic acid may be depleted by cleavage of the targetnucleic acid at one or more locations within the target nucleic acid byone or more nucleic acid guided nuclease(s) in which the nucleasecomponent(s) have nuclease activity. The non-depleted nucleic acidspresent in the initial collection may then be used in a downstreamapplication of interest, such as nucleic acid amplification, nucleicacid sequencing, gene expression analysis (e.g., by array hybridization,quantitative RT-PCR, massively parallel sequencing, etc.), or any otherdownstream application of interest.

Alternatively, a target nucleic acid may be depleted by removal (andoptionally, recovery) of the target nucleic acid from the initialcollection of nucleic acids. As described in more detail below, incertain aspects, removal of a target nucleic acid from the initialcollection is effected using a nucleic acid guided nuclease thatincludes a cleavage-deficient nuclease, which nuclease may include aheterologous component (e.g., a tag) that facilitates removal of thenuclease (and accordingly, the target nucleic acid to which the nucleaseis bound) from the initial collection of nucleic acids. Byremoving/recovering the target nucleic acids from the initialcollection, a subsequent collection of nucleic acids enriched for thetarget sequences may be obtained. This enriched sample (e.g., anexome-enriched sample, a sample enriched for a panel of genes ofinterest, etc.) may then be used in a downstream application ofinterest, such as nucleic acid amplification, nucleic acid sequencing,gene expression analysis (e.g., by array hybridization, quantitativeRT-PCR, massively parallel sequencing, etc.), or any other downstreamapplication of interest.

According to certain embodiments, depleting target nucleic acids presentin the initial collection includes depleting certain species of targetnucleic acids by cleavage of such target nucleic acids, and depletingcertain other species of target nucleic acids by removal (andoptionally, recovery) of such target nucleic acids from the initialcollection of nucleic acids.

A target nucleic acid may vary. By “nucleic acid” is meant a polymer ofany length, e.g., 10 bases or longer, 20 bases or longer, 50 bases orlonger, 100 bases or longer, 500 bases or longer, 1000 bases or longer,2000 bases or longer, 3000 bases or longer, 4000 bases or longer, 5000bases or longer, 10,000 bases or longer, 50,000 or more bases composedof nucleotides, e.g., ribonucleotides or deoxyribonucleotides. In someinstances, the length of the nucleic acids is 100,000 bases or less,e.g., 75,000 bases or less, including 50,000 bases or less, e.g., 25,000bases or less, such as 10,000 bases or less, 5,000 bases or less, 2,000bases or less, 1,000 bases or less, or 500 bases or less.

Depleting a target nucleic acid from the initial collection partiallyreduces, if not completely eliminates, the presence of the targetnucleic acid in the collection. In some instances, the copy number of agiven target nucleic acid in the initial collection of nucleic acids isreduced by 5% or more, such as 10% or more, e.g., 25% or more, including50%, 75%, 90% or more, including embodiments where the presence of thetarget nucleic acid is completely eliminated. Depleting a target nucleicacid reduces the percentage of the target nucleic acid in a sample withrespect to the total nucleic acid in the sample. In certain aspects,after depletion of the target nucleic acid, the percent remaining of thetarget nucleic acid as compared to the initial amount of target nucleicacid in the sample is 50%, 40%, 30%, 25%, 20%, 19%, 18%, 17%, 16%, 15%,14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less,including 0.5%, 0.1%, 0.01% or less. By depleting a target nucleic acidin the initial collection, a type of nucleic acid (e.g., a desirablenucleic acid) may be enriched in the collection. According to certainembodiments, in a sample in which a target nucleic acid has beendepleted, a type of nucleic acid (e.g., DNA, mRNA, microRNA (miRNA),and/or the like) is enriched in the sample such that the percentage ofthe type of nucleic acid remaining in the sample relative to the totalis 5% or more, such as 10% or more, 25% or more, 30% or more, 40% ormore, 50% or more, 60% or more, 70% or more, 75% or more, including 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, and 99.5% or more.

The initial collection of nucleic acids is contacted with a nucleic acidguided nuclease. As used herein, a “nucleic acid guided nuclease” is anassociation (e.g., a complex) that includes a nuclease component and anucleic acid guide component. The nucleic acid guided nuclease may havenuclease/cleavage activity, e.g., catalyzes the hydrolysis of a targetnucleic acid (e.g., a target DNA, a target RNA, etc.) into two or moreproducts thereby depleting the target nucleic acid. Cleavage productsmay be removed from the sample if desired (e.g., to purify the remainingcollection of nucleic acids). In certain embodiments, when it isdesirable to deplete certain target nucleic acids by cleavage, thenumber of distinct nucleic acid guided nucleases used and/or thelocation(s) of the target nucleic acid cleaved by the nucleic acidguided nuclease(s) may be selected such that all or nearly all of thetarget nucleic acid fragments are sufficiently small to be removed bynucleic acid purification steps such as ethanol or isopropanolprecipitation, spin column purification (e.g., using NucleoSpin®Clean-Up columns by Clontech Laboratories, Inc. (Mountain View, Calif.),Solid Phase Reversible Immobilization (SPRI) beads, or the like. Whenthe nuclease component has nuclease activity, the nuclease may generatedouble-stranded breaks in the target nucleic acid, or the nuclease maybe a nuclease that introduces a break in a single strand of adouble-stranded target nucleic acid (e.g., the nuclease component may bea nickase).

In certain aspects, the nuclease is a modified nuclease that does nothave nuclease activity (e.g., is cleavage deficient) as a result of themodification. Such nucleases may be employed to deplete the targetnucleic acid, e.g., upon removal of the target nucleic acid present in acomplex formed between the nucleic acid guided nuclease and the targetnucleic acid, from the initial collection of nucleic acids, which incertain aspects is facilitated by a tag (e.g., an epitope tag) providedon the nuclease.

Any suitable nuclease component may be employed by a practitioner of thesubject methods. The nuclease component may be a wild-type enzyme thatexhibits nuclease activity, or a modified variant thereof that retainsits nuclease activity. In other aspects, the nuclease component may be anon-nuclease protein operatively linked to a heterologous nuclease (or“cleavage”) domain, such that the protein is capable of cleaving thetarget nucleic acid by virtue of being linked to the nuclease domain.Suitable cleavage domains are known and include, e.g., the DNA cleavagedomain of the FokI restriction endonuclease. For example, in certainaspects, the nuclease component of a nucleic acid guided nuclease may bea Cas9 (e.g., a wild-type Cas9 or cleavage deficient Cas9) or othernuclease operably linked to a cleavage domain, such as a FokI cleavagedomain. According to certain embodiments, the nuclease is a mutant thatis cleavage deficient—e.g., Sp, a Cas9 D10A mutant, a Cas9 H840A mutant,a Cas9 D10A/H840A mutant (see, e.g., Sander & Joung (2014) NatureBiotechnology 32:347-355 doi:10.1038/nbt.2842), or any other suitablecleavage deficient mutant. Such a strategy has been successfullyemployed to confer nuclease activity upon zinc finger andtranscription-activator-like effector (TALE) proteins to generate zincfinger nucleases and TALENs, respectively, for genomic engineeringpurposes (see, e.g., Kim et al. (1996) PNAS 93(3):1156-1160, and USPatent Application Publication Numbers US2003/0232410, US2005/0208489,US2006/0188987, US2006/0063231, and US2011/0301073).

According to certain embodiments, the nuclease domain is derived from anendonuclease. Endonucleases from which a nuclease/cleavage domain can bederived include, but are not limited to: a Cas nuclease (e.g., a Cas9nuclease), an Argonaute nuclease (e.g., Tth Ago, mammalian Ago2, etc.),S1 Nuclease; mung bean nuclease; pancreatic DNase I; micrococcalnuclease; yeast HO endonuclease; a restriction endonuclease; a homingendonuclease; and the like; see also Mishra (Nucleases: MolecularBiology and Applications (2002) ISBN-10: 0471394610). In certainaspects, the nuclease component of the nucleic acid guided nuclease is aCas9 nuclease of Francisella novicida (or any suitable variant thereof),which uses a scaRNA to target RNA for degradation (see Sampson et al.(2013) Nature 497:254-257).

As described above, according to certain embodiments, the nucleic acidguided nuclease includes a CRISPR-associated (or “Cas”) nuclease. TheCRISPR/Cas system is an RNA-mediated genome defense pathway in archaeaand many bacteria having similarities to the eukaryotic RNA interference(RNAi) pathway. The pathway arises from two evolutionarily (and oftenphysically) linked gene loci: the CRISPR (clustered regularlyinterspaced short palindromic repeats) locus, which encodes RNAcomponents of the system; and the Cas (CRISPR-associated) locus, whichencodes proteins.

There are three types of CRISPR/Cas systems which all incorporate RNAsand Cas proteins. The Type II CRISPR system carries out double-strandbreaks in target DNA in four sequential steps. First, two non-codingRNAs (the pre-crRNA array and tracrRNA), are transcribed from the CRISPRlocus. Second, tracrRNA hybridizes to the repeat regions of thepre-crRNA and mediates the processing of pre-crRNA into mature crRNAscontaining individual spacer sequences. Third, the mature crRNA:tracrRNAcomplex directs Cas9 to the target DNA via Watson-Crick base-pairingbetween the spacer on the crRNA and the protospacer on the target DNAnext to the protospacer adjacent motif (PAM), an additional requirementfor target recognition. Finally, Cas9 mediates cleavage of target DNA tocreate a double-stranded break within the protospacer.

CRISPR systems Types I and III both have Cas endonucleases that processthe pre-crRNAs, that, when fully processed into crRNAs, assemble amulti-Cas protein complex that is capable of cleaving nucleic acids thatare complementary to the crRNA. In type II CRISPR/Cas systems, crRNAsare produced by a mechanism in which a trans-activating RNA (tracrRNA)complementary to repeat sequences in the pre-crRNA, triggers processingby a double strand-specific RNase III in the presence of the Cas9protein. Cas9 is then able to cleave a target DNA that is complementaryto the mature crRNA in a manner dependent upon base-pairing between thecrRNA and the target DNA, and the presence of a short motif in the crRNAreferred to as the PAM sequence (protospacer adjacent motif).

The requirement of a crRNA-tracrRNA complex can be avoided by use of anengineered fusion of crRNA and tracrRNA to form a “single-guide RNA”(sgRNA) that comprises the hairpin normally formed by the annealing ofthe crRNA and the tracrRNA. See, e.g., Jinek et al. (2012) Science337:816-821; Mali et al. (2013) Science 339:823-826; and Jiang et al.(2013) Nature Biotechnology 31:233-239. The sgRNA guides Cas9 to cleavetarget DNA when a double-stranded RNA:DNA heterodimer forms between theCas-associated RNAs and the target DNA. This system, including the Cas9protein and an engineered sgRNA containing a PAM sequence, has been usedfor RNA guided genome editing with editing efficiencies similar to ZFNsand TALENs. See, e.g., Hwang et al. (2013) Nature Biotechnology 31(3):227.

According to certain embodiments, the nuclease component of the nucleicacid guided nuclease is a CRISPR-associated protein, such as a Casprotein. Non-limiting examples of Cas proteins include Cas1, Cas1B,Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 andCsx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2,Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2,Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2,Csf3, Csf4, homologues thereof, or modified versions thereof. In certainaspects, the nuclease component of the nucleic acid guided nuclease isCas9. The Cas9 may be from any organism of interest, including but notlimited to, Streptococcus pyogenes (“spCas9”, Uniprot Q99ZW2) having aPAM sequence of NGG; Neisseria meningitidis (“nmCas9”, Uniprot C6S593)having a PAM sequence of NNNNGATT; Streptococcus thermophilus (“stCas9”,Uniprot Q5M542) having a PAM sequence of NNAGAA, and Treponema denticols(“tdCas9”, Uniprot M2B9U0) having a PAM sequence of NAAAAC. An examplenucleic acid guided nuclease that includes a Cas9 nuclease and an sgRNAguide component, in which the sgRNA guide component is aligned with acomplementary region of a generalized target nucleic acid, isschematically illustrated in FIG. 1.

In certain aspects, the nuclease component of the nucleic acid guidednuclease is an Argonaute (Ago) nuclease. Ago proteins are a family ofevolutionarily conserved proteins central to the RNA interference (RNAi)platform and microRNA (miRNA) function and biogenesis. They are bestknown as core components of the RNA-induced silencing complex (RISC)required for small RNA-mediated gene regulatory mechanisms. Inpost-transcriptional gene silencing, Ago guided by a small RNA (e.g.,siRNA, miRNA, piRNA, etc.) binds to the complementary transcripts viabase-pairing and serve as platforms for recruiting proteins tofacilitate gene silencing.

Mammals have eight Argonaute proteins, which are divided into twosubfamilies: the Piwi clade and the Ago clade. Of the wild-type Agoproteins (Ago1-4, or EIF2C1-4), only Ago2 has endonuclease activity. Thecrystal structure of full-length human Ago2 (Uniprot Q9UKV8) has beensolved. See, e.g., Elkayam et al. (2012) Cell 150(1):100-110. Similar tothe bacteria counterpart, human Ago2 is a bilobular structure comprisingthe N-terminal (N), PAZ, MID, and PIWI domains. The PAZ domain anchorsthe 3′end of the small RNAs and is dispensable for the catalyticactivity of Ago2. However, PAZ domain deletion disrupts the ability ofthe non-catalytic Agos to unwind small RNA duplex and to form functionalRISC.

When the nuclease component of the nucleic acid guided nuclease is anAgo nuclease, the nuclease may be an Ago nuclease that cleaves DNAduplexes, RNA duplexes, or DNA-RNA duplexes. The Ago nuclease may bederived from any suitable organism, such as a prokaryotic or eukaryoticorganism. In certain aspects, the Ago is a prokaryotic Ago. ProkaryoticAgos of interest include, but are not limited to, Thermus thermophilesAgo (“Tth Ago”), such as the Tth Ago nucleases described in Wang et al.(2008) Nature 456(7224):921-926; and Wang et al. (2009) Nature461(7265):754-761. DNA-guided DNA interference in vivo using Tth Ago and5′-phosphorylated DNA guides of from 13-25 nucleotides in length wasrecently described by Swarts et al. (2014) Nature 507:258-261.

The nucleic acid guided nuclease may include a nuclease having nucleaseactivity (e.g., catalyzes the hydrolysis of a target nucleic acid (e.g.,a target DNA, a target RNA, etc.)), or may be a modified nuclease thatdoes not have nuclease activity (e.g., is cleavage deficient) as aresult of the modification. In some instances, the nuclease component(e.g., a Cas nuclease component) is a cleavage deficient mutant and themethod results in the production of a product composition comprisingtarget nucleic acid/nucleic acid guided nuclease complexes. When part ofthe resultant complex, the target nucleic acid is no longer free in thecollection of nucleic acids, and therefore has been depleted from theinitial collection of nucleic acids. In other aspects, the nucleasecomponent of the complex is a cleavage competent nuclease, but thenucleic acid guided nuclease remains bound to a fragment of the targetnucleic acid subsequent to cleavage of the target nucleic acid. In someinstances, the method further includes separating (e.g., removing)target nucleic acid/nucleic acid guided nuclease complexes (e.g.,including a cleavage competent nuclease component and/or a cleavagedeficient nuclease component such as a D10A Cas9 mutant, a H840A Cas9mutant, a D10A/H840A Cas9 mutant, and/or the like) from otherconstituents of the product composition. Where desired, the nucleasecomponent may include a tag, e.g., an epitope tag, FLAG tag, HA tag, Histag, Myc tag, S-tag, SBP tag, Softag, GST tag, GFP tag, biotin,streptavidin, 6-His tag, etc., e.g., to facilitate separation of thecomplexes (e.g., by affinity purification) from the other components ofthe initial collection.

According to certain embodiments, when the method involves the formationof target nucleic acid/nucleic acid guided nuclease complexes, themethod further includes recovering the target nucleic acids from thecomplexes. Any suitable strategy for recovering the target nucleic acidsmay be employed. Such strategies may include separating the complexesfrom other constituents of the composition, and then disassociating thetarget nucleic acids from the nucleic acid guided nucleases. In certainaspects, the nuclease component of the nucleic acid guided nucleaseincludes a tag (e.g., an epitope tag), and the complexes may beseparated from other constituents by affinity purification. For example,the complexes may be immobilized on the surface of a solid phase (e.g.,a column, a plate, beads (e.g., agarose or magnetic beads), and/or thelike) that includes a binding partner of the tag (e.g., an antibody orother suitable binding partner that binds the tag), and then washed toremove any residual constituents of the composition. The target nucleicacids may then be recovered from the nucleic acid guided nucleases usinga suitable elution buffer (e.g., a buffer that includes a proteindenaturation agent, such as sodium dodecyl sulfate (SDS)), using abuffer that includes a reagent that digests the nuclease component(e.g., proteinase K), using heat denaturation, and/or the like, todisrupt the interactions between the target nucleic acids and thenucleic acid guided nuclease. Approaches for affinity purification andrecovering nucleic acids from protein complexes are described, e.g., inMethods for Affinity-Based Separations of Enzymes and Proteins(Munishwar Nath Gupta, ed., Birkhäuser Verlag, Basel-Boston-Berlin,2002); Chromatin Immunoprecipitation Assays: Methods and Protocols(Collas, ed., 2009); and The Protein Protocols Handbook (Walker, ed.,2002). If desired, the separated target nucleic acids may be furtherpurified by alcohol precipitation, column purification, or any otherconvenient nucleic acid purification strategy.

As summarized above, in certain aspects, the nucleic acid guidednuclease includes a nucleic acid guide component. Any suitable nucleicacid guide component capable of guiding the nuclease component to thetarget nucleic acid may be employed. The nucleic acid guide componentmay be single-stranded or double-stranded as appropriate for theparticular nuclease component employed.

The nucleic acid guide component may be one or more nucleic acidpolymers of any suitable length. In certain aspects, the nucleic acidguide component is a nucleic acid polymer (e.g., a single- ordouble-stranded RNA or DNA) of from 10 to 200 nucleotides in length,such as from 10 to 150 nucleotides in length, including from 10 to 100,from 10 to 90, from 10 to 80, from 10 to 70, from 10 to 60, from 10 to50, from 10 to 40, from 10 to 30, from 10 to 25, from 10 to 20, or from10 to 15 nucleotides in length.

At least a portion of the nucleic acid guide component is complementary(e.g., 100% complementary or less than 100% complementary) to at least aportion of a target nucleic acid of interest. The sequence of all or aportion of the nucleic acid guide component may be selected by apractitioner of the subject methods to be sufficiently complementary toa target nucleic acid of interest to specifically guide the nucleasecomponent to the target nucleic acid. The nucleic acid sequences oftarget nucleic acids of interest are readily available from resourcessuch as the nucleic acid sequence databases of the National Center forBiotechnology Information (NCBI), the European Molecular BiologyLaboratory-European Bioinformatics Institute (EMBL-EBI), and the like.By way of example, when the target nucleic acid(s) of interest is one orboth of human 18S rRNA (1.9 kb) and/or human 28S rRNA, the nucleotidesequences of the 18S rRNA are readily available as those of NCBIreference sequences NR_003286.2 and NR_003287.2, respectively.

Once a target nucleic acid is selected, and based on the availablesequence information for the target nucleic acid, a nucleic acid guidecomponent may be designed such that all or a portion of the nucleic acidguide component is sufficiently complementary to a target region of thetarget nucleic acid to specifically guide the nucleic acid guidednuclease under hybridization conditions to the target region of thetarget nucleic acid, e.g., for cleavage at the target region by thenuclease to deplete the target nucleic acid.

“Hybridization conditions” may include conditions in which the nucleicacid guide component specifically hybridizes to a target region of thetarget nucleic acid, interactions between the target nucleic acid andnuclease component, or both. Whether a nucleic acid guide componentspecifically hybridizes to a target nucleic acid is determined by suchfactors as the degree and length of complementarity between the nucleicacid guide component and the target nucleic acid, and the temperature atwhich the hybridization/contacting occurs, which may be informed by themelting temperature (T_(M)) of the region of the nucleic acid guidecomponent that is complementary to the target region of the targetnucleic acid. The melting temperature refers to the temperature at whichhalf of the nucleic acid guide component-target nucleic acid duplexesremain hybridized and half of the duplexes dissociate into singlestrands. The T_(m) of a duplex may be experimentally determined orpredicted using the following formula T_(m)=81.5+16.6(log₁₀[Na⁺])+0.41(fraction G+C)−(60/N), where N is the chain length and [Na⁺] is lessthan 1 M. See Sambrook and Russell (2001; Molecular Cloning: ALaboratory Manual, 3^(rd) ed., Cold Spring Harbor Press, Cold SpringHarbor N.Y., Ch. 10). Other more advanced models that depend on variousparameters may also be used to predict T_(m) of nucleic acid guidecomponent-target nucleic acid duplexes depending on varioushybridization conditions. Approaches for achieving specific nucleic acidhybridization may be found in, e.g., Tijssen, Laboratory Techniques inBiochemistry and Molecular Biology-Hybridization with Nucleic AcidProbes, Part I, chapter 2, “Overview of principles of hybridization andthe strategy of nucleic acid probe assays,” Elsevier (1993).

According to certain embodiments, the nucleic acid guide component is anRNA guide component (or “guide RNA”). The RNA guide component mayinclude one or more RNA molecules. For example, the RNA guide componentmay include two separately transcribed RNAs (e.g., a crRNA and atracrRNA) which form a duplex that guides the nuclease component (e.g.,Cas9) to the target nucleic acid. In other aspects, the RNA guidecomponent is a single RNA molecule, which may correspond to a wild-typesingle guide RNA, or alternatively, may be an engineered single guideRNA. According to certain embodiments, the nucleic acid guide componentis an engineered single guide RNA that includes a crRNA portion fused toa tracrRNA portion, which single guide RNA is capable of guiding anuclease (e.g., Cas9) to the target nucleic acid.

In certain aspects, the nucleic acid guide component is a DNA guidecomponent, e.g., a single-stranded or double-stranded guide DNA.According to certain embodiments, the guide DNA is phosphorylated at oneor both ends. For example, the guide DNA may be a 5′-phosphorylatedguide DNA oligonucleotide of any suitable length (e.g., any of thelengths set forth above, including for example, from 10 to 30nucleotides in length). The present inventors have demonstrated thatnucleic acid guided nucleases that include such phosphorylated guide DNAoligonucleotides and Tth Ago efficiently deplete a target nucleic acidof interest from an initial collection of nucleic acids based oncomplementarity between the guide DNA oligonucleotide and the targetnucleic acid of interest (see, e.g., the Examples section herein).

As summarized above, the methods of the present disclosure includecontacting an initial collection of nucleic acids with a nucleic acidguided nuclease specific for the target nucleic acid of interest in amanner sufficient to deplete the target nucleic acid from the initialcollection. In certain aspects, contacting the initial collection ofnucleic acids with a nucleic acid guided nuclease includes combining ina reaction mixture the initial collection of nucleic acids, a nucleicacid guide component, and a nuclease component. The nucleic acid guidecomponent and the nuclease component may be stably associated (e.g., asa complex) prior to being added to the reaction mixture, or thesecomponents may be added separately for subsequent association with eachother and targeting/depletion of the target nucleic acid. In certainaspects, at least a portion of the contacting step occurs underconditions in which the nuclease component is active and able to cleavethe target nucleic acid.

The conditions under which the initial collection of nucleic acids iscontacted with the nucleic acid guided nuclease may vary. For example,the conditions may include a temperature at which the nucleic acid guidecomponent specifically hybridizes to the target nucleic acid, such asfrom 0° C. to 10° C. (e.g., 4° C.), from 10° C. to 20° C. (e.g., 16°C.), from 20° C. to 30° C. (e.g., 25° C.), from 30° C. to 40° C. (e.g.,37° C.), from 40° C. to 50° C., from 50° C. to 60° C., from 60° C. to70° C., or from 70° C. to 80° C. Factors and approaches for achievingspecific hybridization between the nucleic acid guide component and thetarget nucleic acid are described hereinabove. In certain aspects,nucleic acids of the initial collection of nucleic acids are denatured(e.g., heat-denatured) to generate single-stranded nucleic acids priorto the contacting step to facilitate hybridization of the nucleic acidguide component to the target nucleic acid.

According to embodiments in which the nuclease component cleaves thetarget nucleic acid, the contacting conditions may include a temperatureat which the particular nuclease employed is active, e.g., has nucleaseactivity. Such temperatures may vary, and in certain aspects includetemperatures from 0° C. to 10° C. (e.g., 4° C.), from 10° C. to 20° C.(e.g., 16° C.), from 20° C. to 30° C. (e.g., 25° C.), from 30° C. to 40°C. (e.g., 37° C.), from 40° C. to 50° C., from 50° C. to 60° C., from60° C. to 70° C., or from 70° C. to 80° C.

The nuclease activity of certain nucleases depends on the presence ofone or more cofactors. When such a nuclease component is employed topractice the subject methods, the contacting conditions may includeproviding any necessary cofactors to the reaction mixture. In certainaspects, the cofactor(s) is one or more divalent cations, such as Mg²⁺,Mn²⁺, Ca²⁺, and/or the like.

The reaction mixture may include one or more buffers (e.g., a Trisbuffer, a PBS buffer, or the like) to ensure that the contacting occursa suitable pH, e.g., at which the nuclease exhibits nuclease activity.For example, the contacting conditions may include the pH of thereaction mixture being from pH 4.5 to 8.5, such as from 4.5 to 5.5, from5.5 to 6.5, from 6.5 to 7.5, or from 7.5 to 8.5.

The contacting step may be performed such that the final concentrationsof the initial collection of nucleic acids, the nucleic acid guidecomponent, and the nuclease component are suitable to deplete the targetnucleic acid. For example, the final concentration of the initialcollection of nucleic acids may be from 0.1 pg/μl to 10 μg/μl, the finalconcentration of the nucleic acid guide component may be from 25 pM to50 μM, and the final concentration of the nuclease component may be from25 pM to 50 μM.

Aspects of the invention include methods of making a nucleic acid guidednuclease, e.g., any of the nucleic acid guided nucleases describedelsewhere herein. Approaches for making the nucleic acid guided nucleasemay vary. In certain aspects, the methods include expressing a nucleicacid guide component and a nuclease component from the same or differentexpression plasmids. Plasmids and associated protocols for expressing anucleic acid guide component and/or a nuclease component arecommercially available and include, e.g., the GeneArt® CRISPR nucleasevectors (Life Technologies, Carlsbad, Calif.).

According to certain embodiments, the present disclosure providesPCR-based methods of producing a nucleic acid guide component specificfor a target nucleic acid of interest. One embodiment of such methods isschematically illustrated in FIG. 2. As shown, a user may design anoligonucleotide primer (shown here as the forward “F” primer) thatincludes: a sequence complementary to a promoter sequence of interest(e.g., a T7, U6, T3 or other promoter); a sequence complementary to ansgRNA guide sequence specific for a target nucleic acid of interest(e.g., specific for a target cDNA transcribed from an rRNA); and asequence complementary to at least a portion of an sgRNA scaffoldsequence. This forward primer may be used in conjunction with anoligonucleotide primer (shown here as the reverse “R” primer) having asequence complementary to at least a portion of the sgRNA scaffoldsequence and any other useful sequences (e.g., a poly dT tract) forproducing an sgRNA. PCR amplification using these primers and a templatethat includes the scaffold and any other desirable elements produces atemplate (designated in FIG. 2 as the “DNA template of sgRNA”) fromwhich a particular sgRNA may be transcribed by in vitro transcriptionand employed in conjunction with a nuclease for, e.g., selectivedepletion of a target nucleic acid according to the methods of thepresent disclosure. Shown in FIG. 3 are example forward (T7-T1-AcGFP,SEQ ID NO: 41; and T7-T2-AcGFP, SEQ ID NO: 42) and reverse (T7-Rev, SEQID NO: 43) oligonucleotides for producing templates from which sgRNAsmay be produced by in vitro transcription, e.g., according to theembodiment shown in FIG. 2. The sequence outlined with dashed rectanglesis the T7 promoter sequence. Underlined sequences are 20 bp crRNAsequences. A polyA sequence is outlined with a solid rectangle.

The initial collection of nucleic acids may vary. Examples of initialcollection of nucleic acids of interest include collections ofdouble-stranded nucleic acids (e.g., double-stranded DNA), collectionsof single stranded nucleic acids (e.g., single-stranded RNA or DNA),mixed collections of double and single stranded nucleic acids, etc. Thecomplexity of the initial collection may also vary, where in someinstances the collection includes 5 or more, 10 or more, 25 or more, 50or more, 100 or more, 250 or more, 500 or more, 1000 or more, 5,000 ormore, including 10,000, 100,000 or more, 500,000 or more, 1 million ormore, 100 million or more, or 1 billion or more distinct nucleic acidsof differing sequence. The initial collection of nucleic acids mayinclude deoxyribonucleic acids, ribonucleic acids, or mixtures thereof.

In certain aspects, the initial collection of nucleic acids of interestis a collection of nucleic acids (e.g., undesired and desired nucleicacids) isolated from a nucleic acid source of interest, including butnot limited to, a nucleic acid sample isolated from a single cell, aplurality of cells (e.g., cultured cells), a tissue, an organ, or anorganism, e.g., bacteria, yeast, or a collection of organisms (such as ametagenomic sample, e.g., sea water containing multiple organisms, afecal sample containing many distinct bacterial species, a buccal swab,etc.), or the like. The term “sample”, as used herein, relates to amaterial or mixture of materials, typically, although not necessarily,in liquid form, containing nucleic acids and/or proteins which onedesires to deplete from an initial collection (e.g., by cleavage orremoval as described elsewhere herein). In certain aspects, the nucleicacid sample is isolated from a cell(s), tissue, organ, and/or the likeof a mammal (e.g., a human, a rodent (e.g., a mouse), or any othermammal of interest). In other aspects, the nucleic acid sample isisolated from a source other than a mammal, such as bacteria, yeast,insects (e.g., drosophila), amphibians (e.g., frogs (e.g., Xenopus)),viruses, plants, or any other non-mammalian nucleic acid sample source.According to certain embodiments, the initial collection of nucleicacids of interest is not genomic DNA.

Any convenient protocol for isolating nucleic acids from such sources,as well as reagents and kits designed for isolating nucleic acids fromsuch sources, may be employed. For example, kits for isolating nucleicacids from a source of interest—such as the NucleoSpin®, NucleoMag® andNucleoBond® genomic DNA or RNA isolation kits by Clontech Laboratories,Inc. (Mountain View, Calif.)—are commercially available. In certainaspects, the nucleic acid is isolated from a fixed biological sample,e.g., formalin-fixed, paraffin-embedded (FFPE) tissue. Nucleic acidsfrom FFPE tissue may be isolated using commercially available kits—suchas the NucleoSpin® FFPE DNA or RNA isolation kits by ClontechLaboratories, Inc. (Mountain View, Calif.).

According to certain embodiments, the initial collection of nucleicacids of interest is produced from a precursor collection of nucleicacids of interest. For example, the initial collection of nucleic acidsof interest may be a collection of DNAs (e.g., cDNAs) transcribed from aprecursor collection of nucleic acids of interest (e.g., RNAs). Incertain aspects, the initial collection of nucleic acids of interest isa collection of cDNAs transcribed from a precursor collection of RNAs ofinterest, where the precursor collection of RNAs of interest includemRNAs, miRNAs, rRNAs, and/or the like, and the target nucleic acid to bedepleted is cDNA transcribed from rRNAs present in the precursorcollection of nucleic acids.

Generating a collection of cDNAs of interest from a precursor collectionof RNAs of interest may include carrying out a reverse transcriptionreaction by combining the precursor collection of RNAs of interest witha suitable polymerase, dNTPs, buffer components that establish anappropriate pH, one or more salts (e.g., KCI), one or more metalcofactors (e.g., Mg²⁺ or Mn²⁺), and the like, under conditions suitablefor a polymerase-mediated extension reaction to occur. Other componentsmay be included, such as one or more nuclease inhibitors (e.g., an RNaseinhibitor and/or a DNase inhibitor), one or more additives forfacilitating amplification/replication of GC rich sequences (e.g.,GC-Melt™ reagent (Clontech Laboratories, Inc. (Mountain View, Calif.)),betaine, single-stranded binding proteins (e.g., T4 Gene 32, cold shockprotein A (CspA), and/or the like), DMSO, ethylene glycol,1,2-propanediol, or combinations thereof), one or more molecularcrowding agents (e.g., polyethylene glycol, or the like), one or moreenzyme-stabilizing components (e.g., DTT present at a finalconcentration ranging from 1 to 10 mM (e.g., 5 mM)), and/or any otherreaction mixture components useful for facilitating polymerase-mediatedextension reactions.

Polymerases that find use in generating a collection of cDNAs ofinterest from a precursor collection of RNAs of interest include, butare not limited to, reverse transcriptases, such as a retroviral reversetranscriptase, retrotransposon reverse transcriptase, retroplasmidreverse transcriptases, retron reverse transcriptases, bacterial reversetranscriptases, group II intron-derived reverse transcriptase, andmutants, variants derivatives, or functional fragments thereof. Forexample, the reverse transcriptase may be a Moloney Murine LeukemiaVirus reverse transcriptase (MMLV RT) or a Bombyx mori reversetranscriptase (e.g., Bombyx mori R2 non-LTR element reversetranscriptase). In certain aspect, the polymerase is capable of templateswitching. Template switching polymerases are commercially available andinclude SMARTScribe™ reverse transcriptase and PrimeScript™ reversetranscriptase available from Clontech Laboratories, Inc. (Mountain View,Calif.). In certain aspects, a mix of two or more different polymerasesis added to the reaction mixture, e.g., for improved processivity,proof-reading, and/or the like. In certain aspects, the polymerase(e.g., a reverse transcriptase such as an MMLV RT or a Bombyx mori RT)is present in the reaction mixture at a final concentration of from 0.1to 200 units/μL (U/μL), such as from 0.5 to 100 U/μL, such as from 1 to50 U/μL, including from 5 to 25 U/μL, e.g., 20 U/μL.

In certain aspects, the initial collection of nucleic acids of interestis produced from a precursor collection of nucleic acids of interest byshearing/fragmenting the precursor collection of nucleic acids ofinterest, e.g., when it is desirable to control the size of the nucleicacids in the initial collection of nucleic acids of interest.Shearing/fragmentation strategies include, but are not limited to,passing a precursor collection of nucleic acids of interest one or moretimes through a micropipette tip or fine-gauge needle, nebulizing thesample, sonicating the sample (e.g., using a focused-ultrasonicator byCovaris, Inc. (Woburn, Mass.)), bead-mediated shearing, enzymaticshearing (e.g., using one or more DNA- or RNA-shearing enzymes),chemical based fragmentation, e.g., using divalent cations (e.g., Mg²⁺,Mn²⁺, and/or Zn²⁺), fragmentation buffer (e.g., a high pH buffer),and/or heat, or any other suitable approach for shearing/fragmenting aprecursor collection of nucleic acids of interest to generate a shorterinitial collection of nucleic acids of interest. In certain aspects, theinitial collection of nucleic acids of interest generated byshearing/fragmentation has a length of from 50 to 10,000 nucleotides,from 100 to 5000 nucleotides, from 150 to 2500 nucleotides, from 200 to1000 nucleotides, e.g., from 250 to 500 nucleotides in length, forexample.

According to certain embodiments, the initial collection of nucleicacids of interest includes nucleic acids (e.g., double-stranded DNA,such as double-stranded cDNA) having one or more sequencing adapterconstructs at one or both ends of the nucleic acids. By “sequencingplatform adapter construct” is meant a nucleic acid construct thatincludes at least a portion of a nucleic acid domain (e.g., a sequencingplatform adapter nucleic acid sequence) or complement thereof utilizedby a sequencing platform of interest, such as a sequencing platformprovided by Illumina® (e.g., the HiSeg™, MiSeg™ and/or Genome Analyzer™sequencing systems); Ion Torrent™ (e.g., the Ion PGM™ and/or Ion Proton™sequencing systems); Pacific Biosciences (e.g., the PACBIO RS IIsequencing system); Life Technologies™ (e.g., a SOLiD sequencingsystem); Roche (e.g., the 454 GS FLX+ and/or GS Junior sequencingsystems); or any other sequencing platform of interest. Such an initialcollection of nucleic acids finds use, e.g., when it is desirable todetermine the sequence(s) of nucleic acids present in the initialcollection of nucleic acids using a sequencing platform.

When the initial collection of nucleic acids (e.g., cDNAs) includessequencing platform adapter constructs, the methods of the presentdisclosure find use in depleting one or more subpopulations of targetnucleic acids in the initial collection (e.g., undesirable sequencessuch as cDNAs transcribed from rRNAs or particular subtypes thereof; ordesirable sequences which are depleted by removal from the initialcollection, recovered to produce a sample enriched for the desirablenucleic acids), followed by sequencing the desirable nucleic acids. Onsequencing platforms that utilize adapter sequences at both ends of anucleic acid to be sequenced (e.g., an Illumina®-Ion Torrent™-basedplatform), a single cleavage event by a nucleic acid guided nuclease ina target nucleic acid (e.g., a cDNA transcribed from an rRNA) rendersthe fragmented target nucleic acid invisible to the sequencer.

According to certain embodiments, the sequencing platform adapterconstruct includes a nucleic acid domain selected from: a domain (e.g.,a “capture site” or “capture sequence”) that specifically binds to asurface-attached sequencing platform oligonucleotide (e.g., the P5 or P7oligonucleotides attached to the surface of a flow cell in an Illumina®sequencing system); a sequencing primer binding domain (e.g., a domainto which the Read 1 or Read 2 primers of the Illumina® platform maybind); a barcode domain (e.g., a domain that uniquely identifies thesample source of the nucleic acid being sequenced to enable samplemultiplexing by marking every molecule from a given sample with aspecific barcode or “tag”); a barcode sequencing primer binding domain(a domain to which a primer used for sequencing a barcode binds); amolecular identification domain (e.g., a molecular index tag, such as arandomized tag of 4, 6, or other number of nucleotides) for uniquelymarking molecules of interest to determine expression levels based onthe number of instances a unique tag is sequenced; a complement of anysuch domains; or any combination thereof. In certain aspects, a barcodedomain (e.g., sample index tag) and a molecular identification domain(e.g., a molecular index tag) may be included in the same nucleic acid.

In some instances, the subject methods include contacting the initialcollection of nucleic acids with a plurality (e.g., a “pool” or“library”) of two or more distinct nucleic acid guided nucleases. Thenucleic acid guided nucleases may be distinct in any desired respects.For example, the methods may employ a pool of nucleic acid guidednucleases in which the pool includes a single type of nuclease component(e.g., a nuclease which may have nuclease activity or, alternatively, becleavage deficient) and two or more species of nucleic acid guidecomponents having different nucleotide sequences. The two or morespecies of nucleic acid guide components may be designed such that theresulting different nucleic acid guided nucleases target multipleregions of a single target nucleic acid, target multiple differenttarget nucleic acids, target multiple regions of multiple differenttarget nucleic acids, etc.

Alternatively, or additionally, the plurality of two or more distinctnucleic acid guided nucleases may include two or more types of nucleasecomponents. For example, the methods may employ a pool/library of anydesired combination of nucleases that differ from one another withrespect to the origin of the nuclease (e.g., nucleases from differentprokaryotic and/or eukaryotic species), nucleases that differ from oneanother with respect to nuclease activity (e.g., the pool/library mayinclude one or more nucleases that have nuclease activity, one or morenucleases that are cleavage deficient, one or more nickases, or anycombination thereof), PAM sequence (e.g., the pool/library may includenucleases that utilize guide nucleic acids having different PAMsequences), and any other combination of nuclease components suitablefor depleting one or more target nucleic acids from the initialcollection. As set forth above, a pool of different nuclease componentsmay be used in conjunction with a pool of different nucleic acid guidecomponents to achieve a desired level of depletion of a desired numberof target nucleic acids. The depletion may include cleaving targetnucleic acids present in the initial collection (e.g., one or moreribosomal and/or mitochondrial RNAs), removing target nucleic acids fromthe initial collection (e.g., to produce a nucleic acid sample enrichedfor the target nucleic acids removed from the initial collection), orboth.

As such, aspects of the present disclosure include methods ofselectively depleting a subpopulation of nucleic acids (e.g., rRNA-and/or mtRNA-derived nucleic acids) from an initial collection ofnucleic acids. In such embodiments, the methods may include contactingthe initial collection of nucleic acids with a library of nucleic acidguided nucleases in a manner sufficient to deplete the subpopulationfrom the initial collection, where the library includes two or moredistinct nucleic acid guided nucleases specific for two or more membersand/or multiple regions of a single member of the subpopulation ofnucleic acids. In these embodiments, the size of the library may vary.For example, the library may include 2 or more, 3 or more, 4 or more, 5or more, 10 or more, 25 or more, 100 or more, 500 or more, 1000 or more,10000 or more, 50000 or more, 100000 or more, 500000 or more, or 1million or more distinct nucleic acid guided nucleases, etc. In certainaspects, the library includes two or more, but 1 million or less, 500000or less, 100000 or less, 50000 or less, 10000 or less, 1000 or less, 500or less, 100 or less, 25 or less, 10 or less, 5, 4, or 3 distinctnucleic acid guided nucleases.

Aspects of the present disclosure further include methods of making alibrary of nucleic acid guided nucleases. Such methods may includeproducing a plurality of distinct nucleic acid guide components usingthe PCR-based approach shown in FIG. 2 and described above. In certainembodiments, the methods include combining a plurality of distinct guidenucleic acids with a one or more distinct nucleases in a mannersufficient to produce the library of nucleic acid guided nucleases. Thenuclease may vary, and in some instances is a Cas nuclease (e.g., Cas9)or Ago nuclease, which independently may or may not have cleavageactivity. The size of the produced library may vary, and in someinstances includes 2 or more, 3 or more, 4 or more, 5 or more, 10 ormore, 25 or more, 100 or more, 500 or more, 1000 or more, 10,000 ormore, 50,000 or more, 100,000 or more, 500,000 or more, or 1 million ormore distinct nucleic acid guided nucleases. In certain aspects, theproduced library includes two or more, but 1 million or less, 500,000 orless, 100,000 or less, 50,000 or less, 10,000 or less, 1000 or less, 500or less, 100 or less, 25 or less, 10 or less, 5, 4, or 3 distinctnucleic acid guided nucleases. In some instances, the nucleic acidguides include separate crRNA and tracrRNA, or a component that includesfunctional elements thereof, e.g., an sgRNA. In some instances, themethods include producing the nucleic acid guides, e.g., by expressingthe nucleic acid guides from plasmids encoding the nucleic acid guides,or by PCR and in vitro transcription, such as described in greaterdetail above and shown in FIG. 2. According to certain embodiments, thenucleic acid guides are produced by solid phase synthesis (e.g., as instandard oligonucleotide synthesis).

In some instances, the nucleic acid guided nucleases of the producedlibrary each include a nucleic acid guide component (e.g., a RNA or DNAguide component) and a nuclease component, e.g., a Cas nucleasecomponent (such as Cas9), an Argonaute nuclease component (e.g., TthAgo, Ago2, or the like). The nuclease component may exhibit cleavageactivity or be a cleavage deficient mutant. Where desired, the nucleasecomponent may further include a tag, such as described above.

Also provided by the present disclosure are compositions. In certainaspects, the compositions include a plurality of distinct nucleic acidguided nucleases. The plurality of distinct nucleic acid guidednucleases may include 2 or more, 3 or more, 4 or more, 5 or more, 10 ormore, 25 or more, 100 or more, 500 or more, 1000 or more, 10,000 ormore, 50,000 or more, 100,000 or more, 500,000 or more, or 1 million ormore distinct nucleic acid guided nucleases, etc. In certain aspects,the plurality of distinct nucleic acid guided nucleases includes two ormore, but 1 million or less, 500,000 or less, 100,000 or less, 50,000 orless, 10,000 or less, 1000 or less, 500 or less, 100 or less, 25 orless, 10 or less, 5, 4, or 3 distinct nucleic acid guided nucleases.

According to certain embodiments, the distinct nucleic acid guidednucleases are distinct based on: the nucleic acid guided nucleaseshaving differing nuclease components; and/or the nucleic acid guidednucleases having nucleic acid guide components of differing nucleotidesequence. For example, the distinct nucleic acid guided nucleases maytarget different regions of the same target nucleic acid based on theguide components having differing sequences complementary to differentregions of the same target nucleic acid, and/or the distinct nucleicacid guided nucleases may target different target nucleic acids based onthe guide components having differing sequences complementary to thedifferent target nucleic acids and/or different species of nucleases(e.g., different Cas9 species) with different PAM sequence requirementsso as to broaden the array of target sequences.

The subject compositions may be present in any suitable environment.According to one embodiment, the composition is present in a reactiontube (e.g., a 0.2 mL tube, a 0.6 mL tube, a 1.5 mL tube, or the like) ora well. In certain aspects, the composition is present in two or more(e.g., a plurality of) reaction tubes or wells (e.g., a plate, such as a96-well plate). The tubes and/or plates may be made of any suitablematerial, e.g., polypropylene, or the like. In certain aspects, thetubes and/or plates in which the composition is present provide forefficient heat transfer to the composition (e.g., when placed in a heatblock, water bath, thermocycler, and/or the like), so that thetemperature of the composition may be altered within a short period oftime, e.g., as necessary for a particular enzymatic reaction to occur.According to certain embodiments, the composition is present in athin-walled polypropylene tube, or a plate having thin-walledpolypropylene wells. In certain embodiments it may be convenient for thereaction to take place on a solid surface or a bead, in such case, theinitial collection of nucleic acids or the nucleic acid guidednuclease(s) may be attached to the solid support or bead by methodsknown in the art—such as biotin linkage or by covalent linkage) andreaction allowed to proceed on the support.

Other suitable environments for the subject compositions include, e.g.,a microfluidic chip (e.g., a “lab-on-a-chip device”). The compositionmay be present in an instrument configured to bring the composition to adesired temperature, e.g., a temperature-controlled water bath, heatblock, or the like. The instrument configured to bring the compositionto a desired temperature may be configured to bring the composition to aseries of different desired temperatures, each for a suitable period oftime (e.g., the instrument may be a thermocycler).

The nucleic acid targeted for depletion can be any target nucleic acidselected by a practitioner of the subject methods. According to oneembodiment, the target nucleic acid is an initial RNA (e.g., an rRNA ormtRNA, and not a reverse transcription product of an RNA). In certainaspects, the target nucleic acid is a reverse (DNA) transcriptionproduct of an initial RNA (e.g., an rRNA or mtRNA). The RNA (e.g., theinitial transcribed RNA) may be any type of RNA (or sub-type thereof)including, but not limited to, a ribosomal RNA (rRNA), a mitochondrialRNA (mtRNA), a microRNA (miRNA), a messenger RNA (mRNA), transfer RNA(tRNA), a small nucleolar RNA (snoRNA), a small nuclear RNA (snRNA), along non-coding RNA (IncRNA), a non-coding RNA (ncRNA), a smallinterfering RNA (siRNA), a transacting small interfering RNA (ta-siRNA),a natural small interfering RNA (nat-siRNA), a transfer-messenger RNA(tmRNA), a precursor messenger RNA (pre-mRNA), a small Cajalbody-specific RNA (scaRNA), a piwi-interacting RNA (piRNA), anendoribonuclease-prepared siRNA (esiRNA), a small temporal RNA (stRNA),a signal recognition RNA, a telomere RNA, a ribozyme, and anycombination of RNA types thereof or subtypes thereof. When the targetnucleic acid is a transcription product of an initial RNA, the methodsmay include depleting all types of such transcription products in thesample (e.g., ribosomal RNA, transfer RNA, microRNA, and the like), orone or more particular types of such transcription products. In certainaspects, the target nucleic acid is a transcription product of aribosomal RNA (rRNA) template. The rRNA template in such instances maybe a eukaryotic 28S, 26S, 25S, 18S, 5.8S, 5S rRNA, or any combinationthereof. In other aspects, the rRNA template may be a prokaryotic 23S,16S, 5S rRNA, or any combination thereof. The subject methods find usein depleting RNA transcription products other than those produced fromribosomal RNAs. For example, the target nucleic acid may be atranscription product of a messenger RNA (mRNA), e.g., a highlyexpressed but clinically irrelevant mRNA from a pool of total RNA ormRNA (e.g., a globulin mRNA in a sample of total or polyA⁺ blood RNA).Other types of RNA transcription products may be targeted for depletion,including a mitochondrial RNA (mtRNA), a precursor messenger RNA(pre-mRNA), a micro RNA (miRNA), a transfer RNA (tRNA), and anycombination thereof. The target transcription product may be a productof RNA from a particular organism, such as bacterial RNA or yeast RNA.According to certain embodiments, the target nucleic acid is not genomicDNA.

In certain aspects, the target molecule is a target nucleic acid, andthe target nucleic acid is a deoxyribonucleic acid (DNA), e.g., intronicor inter-geneic DNA when it is desired to enrich a sample for exonic DNA(e.g., to enrich a sample for nucleic acids corresponding to the exomeof a species of interest) by cleaving intronic or inter-geneic DNApresent in the initial collection, or by capturing the exonic sequencesdirectly. In certain aspects, DNA-based plasmids/vectors such as thoseused for in vitro transcription may be targeted for depletion bycleavage, e.g., after completion of an in vitro transcription reactionto enrich a nucleic acid sample for newly transcribed RNA.

When practicing the methods of the present disclosure, the nucleic acidguided nuclease(s) may be designed such that the frequency of cleavageand resulting fragment sizes of a particular target nucleic acid isselected by a practitioner of the subject methods. For example, asdescribed above, two or more nucleic acid guided nucleases that targetdifferent sequences within a target nucleic acid may be used in amultiplex fashion when it is desirable to cleave the target nucleic acidinto 3 or more fragments. The targeted sequences of the target nucleicacid may be chosen to produce fragments of a desired size, e.g.,fragments which are small enough to be removed from the sample using aspin column, alcohol precipitation, and/or the like.

Utility

The subject methods find use in a variety of different applications,e.g., where it is desirable to deplete irrelevant and/or undesiredmolecules from a sample of interest; where it is desirable to depletenucleic acids of interest by removing the nucleic acids of interest fromthe sample for subsequent recovery (thereby producing a sample enrichedfor the nucleic acids of interest; and/or the like. By depleting theirrelevant and/or undesired molecules, or removing desirable moleculesto produce an enriched sample, the complexity of the sample is reducedand the sample is enriched for molecules of interest. When the moleculesof interest are nucleic acids, reduced complexity and enrichment ofnucleic acids of interest may facilitate and/or improve the results ofdownstream applications such as nucleic acid amplification, nucleic acidsequencing, gene expression analysis (e.g., by array hybridization,quantitative RT-PCR, massively parallel sequencing, etc.), thepreparation of pharmaceutical compositions in which a therapeuticnucleic acid of interest is to be included, and any other applicationsin which reduced sample complexity and enrichment of nucleic acids ofinterest is beneficial.

By way of example, certain embodiments of the subject methods includedepleting nucleic acids from a sequencing library. For example, theinitial collection may be a collection of nucleic acids to be sequencedon a sequencing platform of interest (e.g., a high-throughput or “nextgeneration” sequencing platform such as an Illumina®- or IonTorrent®-based sequencing platform), but the collection includesirrelevant/undesirable nucleic acids which may complicate or interferewith obtaining the sequences of nucleic acids of interest (e.g.,research or clinical interest) in the collection. Theirrelevant/undesirable nucleic acids may be reduced or eliminated usingthe methods of the present disclosure, e.g., by nucleic acid guidednuclease-mediated cleavage, or by producing a sequencing sample that isenriched for sequences of interest by removal of such sequences from theinitial collection using the methods of the present disclosure.

In certain aspects, depletion of a target nucleic acid (e.g., anirrelevant/undesirable nucleic acid, such as a nucleic acid derived froman rRNA, an mtRNA, or the like) renders the target nucleic acidinvisible to the sequencing platform. That is, the depleted (e.g.,cleaved) target nucleic acid is no longer suitable for sequencing on thesequencing platform of interest. For example, Illumina®—and IonTorrent®-based sequencing platforms require nucleic acids havingadapters at each end of the nucleic acids. According to certainembodiments, the nucleic acids of the initial collection of nucleicacids include sequencing adapters at each end, where selective depletionof the target nucleic acid (e.g., cDNAs transcribed from rRNA) includescleaving the target nucleic acid into at least two fragments, none ofwhich will include sequencing adapters at each end as required forsequencing on an Illumina®- or Ion Torrent®-based sequencing platform.As such, the target nucleic acid is rendered invisible to the sequencingplatform, thereby reducing the “load” on the sequencing platform and thecomplexity of the sequencing results.

In certain aspects, when the sequencing platform of interest onlyrequires the nucleic acid to include an adapter at a single end of anucleic acid, the target nucleic acid can be rendered unsuitable forsequencing on the platform by, e.g., cleaving the target nucleic acidsuch that the length of most or all of the resulting fragments are tooshort to be sequenced on the sequencing platform. This may beaccomplished by cleaving the target nucleic acid at a single location,or at two or more locations within the target nucleic acid, to generatefragments of insufficient length to be sequenced on the sequencingplatform, e.g., because the platform requires nucleic acids of greaterlength, or the fragments are lost during a purification procedure (e.g.,bead purification, such as SPRI bead-based purification) prior tosequencing. The location(s) to be cleaved and the distance(s) betweencleavage sites may be selected by a practitioner of the subject methodsto generate cleavage fragments of the desired length, e.g., usingavailable nucleic acid sequence information for a target nucleic acid ofinterest and designing nucleic acid guide component(s) with sequencecomplementarity to the selected cleavage site(s). In this way, thetarget nucleic acid is rendered invisible to the sequencing platform andthe load on the sequencing platform and complexity of the sequencingresults is reduced.

As described above, the methods of the present disclosure also find usein selectively recovering one or more types of nucleic acids of interestfrom an initial collection of nucleic acids. For example, in certainaspects, depleting one or more types of target nucleic acids from theinitial collection of nucleic acids includes capturing the targetnucleic acid via formation of target nucleic acid/nucleic acid guidednuclease complexes, and then recovering the target nucleic acids fromthe complexes, e.g., for downstream analysis (e.g., quantitativeanalysis, sequence analysis, and/or the like). Approaches for targetnucleic acid capture and recovery include, e.g., affinity-basedapproaches (which in certain aspects is facilitated by the nucleasecomponent including an epitope/affinity tag), as described hereinabove.Accordingly, the subject methods find use in selectively obtaining oneor more target nucleic acids of interest from collections of nucleicacids, which in certain aspects are complex collections of nucleic acids(e.g., a collection of cDNAs produced by reverse transcription of atotal RNA sample, exons from a genomic library, or any other complexnucleic acid collections of interest).

Kits

Also provided by the present disclosure are kits useful for practicingthe subject methods. The kits may include one or more of any of thecomponents described above in relation to the subject methods andcompositions. For example, the kits may include a primer and a templatefor generating PCR amplification products from which a nucleic acidguide component may be produced by in vitro transcription. Such reagentsmay include any reagents useful in practicing the method for producing anucleic acid guide component (e.g., an sgRNA) shown in FIG. 2 anddescribed hereinabove. According to certain aspects, the kit includes areverse primer for use in conjunction with a forward primer provided bya user of the kit, where at least a portion of the forward primerincludes a nucleic acid sequence complementary to a target nucleic acidselected for depletion by the user.

In some instances, the kits include: a vector that includes a sgRNAscaffold template domain; a reverse primer configured for use with thevector in a PCR reaction; and an RNA polymerase. In some instances, thereverse primer comprises a polyA domain and an sgRNA scaffold domain.While the RNA polymerase may vary, in some embodiments the RNApolymerase is a T7 polymerase. Where desired, the kit further includesone or more of a DNA polymerase; a PCR buffer; a nuclease (e.g., a Cas,Ago, or other nuclease) which may or may not have cleavage activity; acontrol nucleic acid; etc.

Components of the subject kits may be present in separate containers, ormultiple components may be present in a single container. For example,when the kit includes a primer for generating a template DNA from whichan RNA guide component is produced by in vitro transcription, the primermay be provided in a separate container, or in a container that includesa second component of the kit (e.g., a buffer or the like).

In addition to the above-mentioned components, the subject kit mayfurther include instructions for using the components of the kit topractice the subject methods. The instructions for practicing thesubject methods may be recorded on a suitable recording medium. Forexample, the instructions may be printed on a substrate, such as paperor plastic, etc. As such, the instructions may be present in the kits asa package insert, in the labeling of the container of the kit orcomponents thereof (i.e., associated with the packaging or subpackaging)etc. In other embodiments, the instructions are present as an electronicstorage data file present on a suitable computer readable storagemedium, e.g., portable flash drive, DVD, CD-ROM, diskette, etc. In yetother embodiments, the actual instructions are not present in the kit,but means for obtaining the instructions from a remote source, e.g. viathe internet, are provided. An example of this embodiment is a kit thatincludes a web address where the instructions can be viewed and/or fromwhich the instructions can be downloaded. As with the instructions, themeans for obtaining the instructions is recorded on a suitablesubstrate.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 Depletion of a Target PCR Fragment

In this example, a nucleic acid guided nuclease was produced and, asproof of concept, used to deplete a PCR product having a target sequencecorresponding to an rRNA sequence. Using the method outlined in FIG. 2and described above, two distinct sgDNA templates (CMBO640 and CMBO641)were generated by PCR using the Advantage® HD polymerase (ClontechLaboratories, Mountain View, Calif.). The PCR products are seen as themain (lower) bands in the gel image shown in FIG. 4. Between 100-120 ngof the templates were used in an in vitro transcription reaction inaccordance with the Takara T7 RNA polymerase manual to produce thecorresponding sgRNAs.

The in vitro transcribed CMBO640 (72 ng/μl) and CMBO641 (90 ng/μl)sgRNAs were then tested for their ability, when combined with Cas9 (500ng), to cleave a PCR product (260 ng) having a sequence corresponding toan rRNA sequence. The results are provided in FIG. 5. As shown,combining Cas9, the COMBO640 and/or COMBO641 sgRNAs, and the PCR productresults in cleavage of the PCR product.

Example 2 Multiplex Depletion of DNAs Corresponding to Full-Length 18SrRNA

In this example, it is shown that two different sgRNAs having differenttarget sequence specificities can be used in conjunction (i.e., in amultiplex fashion) to deplete a target DNA having a sequencecorresponding to full-length 18S rRNA. As shown in the gel imageprovided in FIG. 6, a nucleic acid guided nuclease that includes Cas9and a first sgRNA (“sgRNA1”, fourth lane from the left) and a nucleicacid guided nuclease that includes Cas9 and a second sgRNA (“sgRNA2”,fifth lane from the left) are separately able to cleave at differentlocations a target DNA having a sequence corresponding to full-length18S rRNA. When the two nucleic acid guided nucleases are both combinedwith the target DNA (sixth lane from the left), the target DNA iscleaved at both of the respective cleavage sites, indicating thatmultiplexed target depletion occurred.

Example 3 Depletion of Undesirable Sequences from Next GenerationSequencing Libraries

In this example, the ability of nucleic acid guided nucleases to depleteunwanted sequences from next generation sequencing libraries was tested.A next generation sequencing library of cDNAs transcribed from humanbrain total RNA was treated with a sgRNA/Cas9 nucleic acid guidednuclease to deplete cDNAs corresponding to 18S rRNA by cleaving atposition 1022-1041 of the cDNAs corresponding to 18S rRNA prior tosequencing the library. The resulting sequences were mapped to the rRNAand the number of reads overlapping 1022-1041 was divided by the totalnumber of reads mapping to rRNA. The ratio was plotted and normalized tocontrol.

As shown in Panel A of FIG. 7, depletion of the target sequence onlyoccurred in the library treated with both Cas9 and the specific guideRNA.

In a separate experiment, a target cDNA corresponding to 18S rRNA wasdegraded using a pool of sgRNAs targeting the target cDNA along itslength about every 50 bp. The pool was generated by PCR amplifying 35sgDNA templates (using the approach shown in FIG. 2), and then in vitrotranscribing the corresponding 35 sgRNAs in a pooled in vitrotranscription reaction. This is one of the key advantages of the methodof the present disclosure shown in FIG. 2, as large quantities of acomplex collection of different sgRNA sequences can be produced in asingle reaction for depleting a single target (by tiling across thetarget with sgRNAs having different target-specific sequences) ordepleting multiple targets using the collection of sgRNAs.

In this experiment, a model target was used that included a targetcorresponding to the full-length 18S RNA (25 ng) and 55 ng of a 5800 bpplasmid iPCR product (used as a surrogate for the 28S rRNA). This ratioapproximates the ratio (in mass) of 18S and 28S rRNA in a 20 nM RNA-Seqlibrary with no depletion. As shown in Panel B of FIG. 7, the 18Sfragment was thoroughly depleted/degraded by treatment with the sgRNApool, while the iPCR fragment was unaffected. Depletion of the targetnucleic acid results in failure of the target to cluster on thesequencer (e.g., an Illumina® sequencer) because, at most, the targetonly has a sequencing adapter at one of its ends following cleavage.Alternatively, the depleted target fragments are sufficiently small thatthey are lost following purification (e.g., SPRI bead purification) andthus not available for sequencing.

Example 4 Depletion of 18s rRNA Using a Pool of Nucleic Acid GuidedNucleases

In this experiment, sequencing libraries were generated from 100 ngHuman Brain Total RNA (Clontech) using the SMARTer Stranded RNA-Seq Kit(Clontech). 70 ng of library was incubated with 0, 2, or 5 μg ofrecombinantly purified Cas9 and 0 or 191 ng of the 35 sgRNA pooldescribed in the previous experiment in 20 μl 1× NEB3.1 buffer for onehour at 37° C. The Cas9 was then heat inactivated for 10 minutes at 70°C. The libraries were then pooled and sequenced on a MiSeq instrument.The resulting sequences were mapped against the human genome, hg19, andrRNA transcripts simultaneously using the STAR aligner. FIG. 8illustrates the reduction in sequence coverage of the 18S rRNAtranscript resulting only from treatment with both Cas9 and the sgRNApool. The number of sequencing reads mapped to the 18S rRNA werenormalized to the total number of reads sequenced and plotted in FIG. 9,demonstrating an ˜75% reduction in sequences mapping to the 18Stranscript.

Examples 1-4 Sequence Information

The sgRNA described in examples 1-4 contained the sgRNA scaffold shownin FIG. 1 with the following target-specific sequences:

sgRNA1 used in examples 1 and 2: (SEQ ID NO: 1) UUAUCAGAUCAAAACCAACCsgRNA2 used in examples 1, 2 and 3: (SEQ ID NO: 2) UAAUCAAGAACGAAAGUCGGpool of 35 sgRNA used in examples 3 and 4: (SEQ ID NO: 3 to 37)GACAAGCAUAUGCUACUGGC CGGCGCAAUACGAAUGCCCC CGGUACAGUGAAACUGCGAACGCUCUGGUCCGUCUUGCGC GGAGAGGAGCGAGCGACCAA UAAUCAAGAACGAAAGUCGGUAGAGCUAAUACAUGCCGAC CGGUCGGCAUCGUUUAUGGU UUAUCAGAUCAAAACCAACCGUUUCCCGGAAGCUGCCCGG GGGGCGGGCGCCGGCGGCUU CUGAAACUUAAAGGAAUUGACGAUCGCACGCCCCCCGUGG GGCUUAAUUUGACUCAACAC GGUAGUCGCCGUGCCUACCACUGUCAAUCCUGUCCGUGUC UCAGGGUUCGAUUCCGGAGA GCAUGGCCGUUCUUAGUUGGUGCGCGCCUGCUGCCUUCCU GCCAGAGUCUCGUUCGUUAU AACAAUACAGGACUCUUUCGGCGUCCCCCAACUUCUUAGA CCUCGUUAAAGGAUUUAAAG UGUUAUUGCUCAAUCUCGGGUAUUGGAGCUGGAAUUACCG AGCGUGUGCCUACCCUACGC AAAGCUCGUAGUUGGAUCUUCCGUUGAACCCCAUUCGUGA CAAGGGGCGGGGACGGGCGG UACUGGGAAUUCCUCGUUCAUCUUAGCUGAGUGUCCCGCG GGCGGUGUGUACAAAGGGCA CAAAGCAGGCCCGAGCCGCCGGCCCUCGGAUCGGCCCCGC GGACCGCGGUUCUAUUUUGU

Example 5 Depletion of Target Nucleic Acids by Argonaute (Ago)

In this example, the ability of the Argonaute (Ago) protein to deplete atarget nucleic acid was assessed. His-tagged Tth Ago was expressed andpurified (FIG. 10, Panel A). Depletion of a 5′ FAM labelled ssDNArepresentative target was carried out by combining the 500 nMsingle-stranded DNA, Tth Ago, and 100 nM 5′ phosphorylated targetingoligonucleotide complementary to the target single-stranded DNA andincubated at 75° C. for one hour. As shown in FIG. 10, Panel B, thesingle stranded target was not cleaved in the absence of Ago (far rightlane), but was cleaved in the presence of Ago and the targetingoligonucleotide at various Ago concentrations. FIG. 11 demonstrates asimilar experiment, but with 20 nM ssDNA target and 50 nM guide DNA tobe representative of depleting common library concentrations.

In Example 5, the guide DNA oligo was: (SEQ ID NO: 38)/5′Phos/TGAGGTAGTAGGTTGTATAGT; and the targeting oligo was:(SEQ ID NO: 39) /5′6-FAM/AGGTGATAAGACTATACAACCTACTACCTCGAATGTCCGT

Example 6 Depletion of Target Nucleic Acids from a Collection byArgonaute (Ago)

As demonstrated in Example 5, Argonaute is capable of depleting targetssDNA molecules in solution. If the desired target is double stranded,the double stranded material may be converted to a single strands bydenaturation, such as with heat or high pH, or by degrading thenon-target strand by exonuclease treatment. For example, the mixture maybe amplified by a common pair of PCR primers, only one of which is 5′phosphorylated. The amplified product could then be treated withA-exonuclease to yield a ssDNA product. This ssDNA product could then beused as a substrate for cleavage by Ago and a targeting oligonucleotide.

Example 7 Recovery of Targeted Sequences

In this Example the 6× HN tagged D10A/H840A mutant of Cas9 (referred toherein as dCas9), was expressed and purified in E. coli (FIG. 12, PanelA). RNA-Seq libraries were generated from 100 ng Human Brain PolyA-plusRNA (Clontech) and the SMARTer Stranded RNA-Seq kit (Clontech). 10 ng oflibrary was combined with ˜1.25 μg dCas9 and 76 ng of a pool of 190sgRNA designed every ˜50 bp on the human 5S, 5.8S, 18S, 28S, mt12S, andmt16S sequences in 10 μl 1× NEB3.1. The mixture was incubated at 37° C.for one hour. Then 10 μg of salmon sperm DNA (Life Technologies) wasadded and the reaction was allowed to proceed for an additional 30minutes at 37° C. 5 μl of TALON magnetic beads (Clontech) equilibratedin 10 μl NEB3 was added to the tubes and incubated at 25° C. withrotation for 30 minutes. The beads were washed twice with 200 μl NEB. 50μl of SeqAmp PCR Mastermix (1× SeqAmp Buffer, 1 μl SeqAmp DNApolymerase, and 250 nM Illumina P5 and P7 PCR primers) was added to thewashed beads and amplified with 15 cycles of PCR (94° C. 1 minute, 15×(98° C.-15 s, 55° C.-15 s, 68° C.-30 s)). The amplified product waspurified with 50 μl AMPure beads (Beckman) according to themanufacturer's instructions and eluted in 20 μl 10 mM Tris-HCl pH8.50.1% Tween-20. The library was then sequenced on a MiSeq instrument. Theresulting sequences were mapped against the human genome, hg19, and rRNAtranscripts simultaneously using the STAR aligner. Reads mapping to rRNAwere identified by Picard RNA-Seq Metrics, and the relative amounts ofsequences mapping to rRNA are plotted in FIG. 12, Panel B, showing a˜340% enrichment verses an untreated library. This example demonstratesthat nucleic acid guided nucleases may be used to deplete target nucleicacids by removal of the target nucleic acids from an initial collectionof nucleic acids, for subsequent recovery and production of a sampleenriched for the target nucleic acids.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims.

What is claimed is:
 1. A method of selectively depleting target cDNAsfrom a sample, the method comprising: obtaining a sample comprising bothtarget cDNAs and non-target cDNAs, wherein the target cDNAs comprisecDNAs transcribed from ribosomal RNAs, contacting the sample with anucleic acid guided nuclease and a guide nucleic acid that guides thenucleic acid guided nuclease to the target cDNAs, wherein the nucleicacid guided nuclease, the guide nucleic acid, and the target cDNAs forma complex in which the guide nucleic acid specifically hybridizes withthe target cDNAs, and selectively depleting the target cDNAs from thesample by cleaving the target cDNAs with the nucleic acid guidednuclease in the complex.
 2. The method according to claim 1, wherein thetarget cDNAs are double-stranded nucleic acids.
 3. The method accordingto claim 1, wherein the target cDNAs are single-stranded nucleic acids.4. The method according to any of claim 1, wherein the guide nucleicacid is RNA.
 5. The method of claim 4, wherein the RNA is single-guideRNA specifically hybridized to human 5S, 5.8S, 18S, or 28S rRNAs.
 6. Themethod according to claim 4, wherein the nucleic acid guided nuclease isa Cas nuclease.
 7. The method according to claim 1, wherein the guidenucleic acid is DNA.
 8. The method according to claim 7, wherein thenucleic acid guided nuclease is a Tth Ago nuclease.
 9. The methodaccording to claim 1, wherein the method comprises further contactingthe sample with one or more additional distinct nucleic acid guidednucleases.
 10. The method of claim 9, wherein the one or more additionaldistinct nucleic acid guided nucleases are guided by one or moredistinct guide nucleic acids.
 11. A method of selectively depletingtarget cDNAs from a sample, the method comprising: obtaining a samplecomprising both target cDNAs and non-target cDNAs, wherein the targetcDNAs comprise cDNAs transcribed from ribosomal RNAs, contacting thesample with a nucleic acid guided nuclease and a guide nucleic acid thatguides the nucleic acid guided nuclease to the target cDNAs, wherein thenucleic acid guided nuclease is a cleavage deficient mutant that doesnot have nuclease activity and comprises a tag, and the nucleic acidguided nuclease, the guide nucleic acid, and the target cDNAs form acomplex in which the guided nucleic acid specifically hybridizes withthe target cDNAs, and separating the complex from the sample byimmobilizing the complex to the surface of a solid phase including abinding partner that binds to the tag, thereby depleting the targetcDNAs from the sample.
 12. The method according to claim 11, wherein thetag is an epitope tag, FLAG tag, HA tag, His tag, Myc tag, S-tag, SBPtag, Softag, GST tag, GFP tag, biotin, streptavidin, or 6-His tag. 13.The method according to any of claim 11, wherein the guide nucleic acidis RNA.
 14. The method according to claim 13, wherein the nucleic acidguided nuclease is a Cas nuclease.
 15. The method according to claim 11,wherein the guide nucleic acid is DNA.
 16. The method according to claim15, wherein the nucleic acid guided nuclease is a Tth Ago nuclease.